Additive Manufacturing of Channels

ABSTRACT

A method is disclosed for 3D printing of soft polymeric material such as a hydrogel or elastomer for scaffolds or devices with embedded channels with tunable shape and size such as a channel inner diameter). The method utilizes extrusion based printing of polymer solutions usually referred as direct ink writing (DIW) or BioPlotting, and requires sequential printing of a photocurable polymer solution, herein, referred as the matrix material, and a sacrificial polymer solution that may dissolve in an aqueous media.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of the filing date of U.S.Provisional Patent Application No. 62/715,869, filed Aug. 8, 2018, thedisclosure of which is hereby incorporated by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to 3D printing. In particular, thepresent disclosure relates to additive manufacturing of 3D scaffolds anddevices with embedded channels for vasculature.

BACKGROUND

Bioink refers generally to materials composed of living cells that canbe used for 3D printing of complex tissue models. Bioinks are materialsthat mimic an extracellular matrix environment to support high cellviability and potentially to support the adhesion, proliferation, anddifferentiation of living cells.

Additive manufacturing, commonly known as 3D printing, has becomeincreasingly popular over recent years. Additive manufacturing refersgenerally to processes used to manufacture a three-dimensional object inwhich successive layers of material are formed under computer control tocreate a 3D construct or a device.

One application of additive manufacturing allows fabrication of complex3D structures from a patient's own medical image, which is not possiblewith conventional fabrication techniques. Additive manufacturing ofbiological materials, i.e., bioinks (cells, cell-laden hydrogels,extracellular matrix materials, and their various combinations), isreferred as bioprinting. Single extrusion-based bioprinting is one ofthe mostly utilized 3D printing approaches for tissue and organ printingstudies. Recent focus in the biomanufacturing field is to fabricate 3Dtissue and organ mimetics, such as in the form of organ-on-a-chipdevices, for disease modeling and drug development and screening, tohuman-scale scaffolds for tissue regeneration.

One of the main bottle necks and problems related to 3D printing forthis application of bioprinting is forming channels within softpolymeric materials, such as hydrogels and elastomers. These channelsare crucial for organ-on-a-chip devices, tissue/disease models andhuman-scale scaffolds/tissue mimetics for perfusion of required solublecomponents as well as development of vascularization.

Recent advances in 3D bioprinting allow development of several printingmethods to overcome this problem, but have been met with limitedsuccess. These methods include gel-casting, free-form printing, andcoaxial printing.

In one method, a sacrificial polymer ink is 3D printed inside a mold,which is then filled with the matrix hydrogel or elastomer, and followedby a crosslinking process. The 3D printed sacrificial structure isusually made of water soluble polymers or hydrogels, such as sugar-basedpolymers or Pluronic F-127, or other sol-gel transition gel, such asagarose. The sacrificial structure is then dissolved leavinginterconnected channels within the hydrogel matrix. In this technique,it is almost impossible to form individual channels spatiallydistributed within the hydrogel.

A second method requires a bath of support material, which allows 3Dprinting of another ink within the support material. This limits theavailable support systems as they should allow a needle to move freelywithin the support material. Support material is usually a highlyviscous polymer solution, a shear thinning hydrogel, or micro sizeparticle/hydrogel suspension.

For free-form printing, there are mainly three approaches. In a firstapproach, the support material within the bath is a sacrificialmaterial, and matrix material, usually a curable hydrogel solution, isprinted inside this sacrificial material followed by the removal of thesacrificial material. For instance, an alginate hydrogel can be printedin a CaCl₂-containing gelatin reservoir at room temperature. After theprinting process, the whole reservoir, including the printed structure,was heated to 37° C. to gradually melt the gelatin support. A secondapproach utilizes self-healing hydrogels and sacrificial hydrogels,which enables either printing a sacrificial hydrogel into theself-healing hydrogel followed by the removal of the sacrificialhydrogel. A third free-form approach utilizes digital-light-processing(DLP) printing technology, which requires a laser to spatially cure aphoto-curable polymer solution within a reservoir.

It is still a challenge to create vascularized scaffolds or hydrogelswith embedded channels for vascularization and soft microfluidic devicesfrom elastomers or hydrogels in a single step. This requires a fast andsimple approach to create channels within soft systems such as hydrogelsand elastomers. Although the above-mentioned additive manufacturing (3Dprinting) techniques allow fabrication of channels within hydrogels orelastomers, these techniques require development of special materialsand are not applicable to a wide range of materials. In addition, theseconventional techniques require the use of excess material, whichincreases the cost of the fabrication. Accordingly, there still remainsa need for a method of making 3D scaffolds and devices without the abovedrawbacks.

SUMMARY

The present disclosure avoids the drawbacks of conventional 3D printingmethods and provides many other advantages. No additional steps arerequired. A reservoir of materials is eliminated. The present method notonly eliminates the need of these items, but also allows direct printingof a matrix material and sacrificial material sequentially. The presentmethod includes fabrication of polymeric scaffolds/devices with embeddedchannels using a novel extrusion based printing approach involvingprinting of the sacrificial polymer/hydrogel within the interface of thephotocurable matrix layer. The printing may be sequential.

The present method utilizes a photo-curable solution of matrix polymermaterial (e.g., cell-laden hydrogel). Matrix polymer material refers tothe material that comprises the main scaffold or device. Also utilizedis a sacrificial polymer/hydrogel that refers to a polymer/hydrogel thatcan be removed after printing, for instance via dissolution in anaqueous solution. Also utilized is an extrusion-based printer system,which allows extrusion of polymer solutions under applied pressure. Adual printing system is used to first print matrix material up to acertain thickness that may be determined by the user.

After each print layer, the matrix material is exposed to light topartially crosslink the printed layer. This allows self-supporting ofthe matrix material enabling printing of low viscous bioinks. Thus, inone embodiment, the method includes the step of sequential printing of aphotocurable polymer solution and a sacrificial polymer solution, inwhich the sacrificial polymer solution is printed directly within theinterface of a partially crosslinked and freshly printed photocurablelayer. When needed or as determined by the operator, the sacrificiallayer is printed directly within the freshly printed matrix polymersolution before light exposure of this particular layer. Depending onthe implementation, the sacrificial layer may be 100-1000 micronsdepending on the desired channel size.

After printing of the sacrificial polymer light is exposed to partiallycross-link the sacrificial polymer layer, and a new layer of matrixmaterial is printed. This process is repeated as needed.Scaffolds/devices may be created in human scale. There is no limit intotal device thickness in the current approach. After the printing isdone, the system is exposed to light (for example 4 minutes, butexposure time can differ for different materials) to fully crosslink theprinted device/scaffold. Then the scaffold/device is immersed in anaqueous solution (such as phosphate buffer solution, PBS) to remove thesacrificial polymer, which will lead to formation of channels.

For easy handling of the scaffold/device, the printing could be done ona surface modified glass slide or microscope cover slip. Surfacemodification is done using 3-(trimethoxysilyl)propyl methacrylate thatallows covalent cross linking of the matrix polymer to the glass slide.Sequential printing or printing of the sacrificial polymer within theinterface of the printed matrix material allows formation of channelsnever before achievable by conventional methods.

In one embodiment, an extrusion-based printer system is disclosed, whichallows extrusion of polymer solutions under applied pressure. Dualprinting is used to first print matrix material up to a certainthickness as determined by the user. Again after each print layer, thematerial is exposed to light to partially crosslink the printed layer.This allows self-supporting of the matrix material.

Furthermore, the present method and system does not require a shearthinning material unlike conventional 3D printing methodologies. Theneedle used in the 3D printing of layers is only within the previouslyprinted interface layer so the material doesn't require shear thinningbehavior for needle to move freely. This feature of the presentdisclosure provides a significant improvement over current 3D printingmethodologies and allows the use of almost any photo-curable material asa matrix material.

The above objects and advantages are met by the presently disclosedmethod and apparatus. In addition the above and yet other objects andadvantages of the present invention will become apparent from thehereinafter-set forth Brief Description of the Drawings, DetailedDescription of the Invention, and claims appended herewith. Thesefeatures and other features are described and shown in the followingdrawings and detailed description.

Furthermore, any combination and/or permutation of the embodiments areenvisioned. Again, other objects and features will become apparent fromthe following detailed description considered in conjunction with theaccompanying drawings. It is to be understood, however, that thedrawings are designed as an illustration only and not as a definition ofthe limits of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

To assist those of skill in the art in making and using the disclosedmethod to make a device using 3D printing and associated systems andmethods, reference is made to the accompanying figures, wherein:

FIG. 1 shows views outlining a printing method to create a channelembedded in a 3D hydrogel, in accordance with one embodiment of thepresent disclosure;

FIG. 2 shows views outlining one embodiment of a post-printing method tofully crosslink the matrix hydrogel and dissolve the sacrificialhydrogel to create channels;

FIGS. 3A-3D show 3D design, and specifically (Top Row) CAD designs ofthe hydrogel scaffolds with channels (light gray: matrix; dark gray:sacrificial). (FIGS. 3A-D) Pictures showing the printed scaffolds afterremoval of the sacrificial regions: (FIG. 3A) channels with varyingchannel diameter, (FIG. 3B) two-layer channel structure (channels are indifferent planes), (FIG. 3C) interconnected elliptical channels, and(FIG. 3D) channels forming NJIT. Coarse channels (500 μm in diameter)are printed for visual clarity and easy access with a regular sizeneedle, but 100 μm channels can be printed;

FIGS. 4A-4C show 3D printed structures, and feasibility studies showing(Left FIG. 4A) a schematic of a channel design, and (Center FIG. 4B) atop view of an actual sample having the channel design of FIG. 4A; 3Dprinted construct with Pluronic doped with red food coloring; and (RightFIG. 4C) confocal images of the channels in FIG. 4B taken at within theregion of the black rectangle in Right and perfused with methacrylatedrhodamine containing PBS, wherein the channels are smooth andcylindrical in shape with width equal to ˜500 μm and scale bars are 500μm and;

FIG. 5 shows a flow chart to create a channel embedded in a 3Dstructure, in accordance with one embodiment of the present disclosure.

DETAILED DESCRIPTION

Vascularization is a major limitation for development of human-scalefunctional tissues or organs. Fundamentally it requires ability tocreate channels within 3D soft scaffolds that mimics human tissue.Creating well defined channels within 3D hydrogels and/or elastomers arealso important for development of soft devices towards organ-on-a-chipsystems, such as but not limited to 3D tissue/disease models, to detectdisease or screen for drugs. The present disclosure addresses this majorgap in creating channels within soft 3D polymeric systems using additivemanufacturing. Although additive manufacturing is utilized to createchannels previously, this present novel approach eliminates the use ofspecially designed printers, specialty shear thinning material, therequirement for multiple steps, and the use of excess materials. Thisnew approach is suitable for any photocurable hydrogel and elastomerformulation with the use of a sacrificial polymer ink such as polymersor hydrogels that are soluble in an aqueous media.

Exemplary embodiments are directed to 3D printing of soft polymericscaffolds or devices. It should be understood that embodiments cangenerally be applied to other scaffolds or devices.

In one embodiment, a method is disclosed for 3D printing of softpolymeric (hydrogel or elastomer) scaffolds or devices with embeddedchannels with tunable shape and size (i.e., channel inner diameter). Themethod utilizes extrusion based printing of polymer solutions usuallyreferred as direct ink writing (DIW) or BioPlotting, and requiressequential printing of a photocurable polymer solution referred hereinas the matrix material, and a sacrificial polymer solution, i.e.,preferable to dissolve in an aqueous media such as phosphate buffersaline (PBS).

In this embodiment, the fabrication process starts with 3D printingseveral layers of matrix material. Matrix material could be anyphotocurable hydrogel ink. The ink is not required to self-supportitself after printing, which allows the use of a wide range ofmaterials. After printing of each layer, the printed matrix solution isexposed to light for a very short time (˜10 s) to partially cure theprinted layer. This allows the matrix hydrogel to self-support itself.When the desired matrix material height (thickness) is reached, oneadditional layer of matrix material is printed but not exposed to light.The sacrificial material is directly printed within this matrix layer.This uncrosslinked matrix layer supports the printed sacrificialpolymer/hydrogel. The system is then exposed to light to partiallycrosslink the matrix layer. Then another layer of matrix material isprinted followed by light exposure. This process is repeated as neededto reach the final desired scaffold/device thickness.

The 3D printed construct is exposed to light to fully crosslink thematrix polymer, such as a hydrogel or elastomer, and immersed in anaqueous media to dissolve the sacrificial polymer or hydrogel.Dissolution of the sacrificial polymer/hydrogel leads to channelformation within the matrix hydrogel or elastomer. This method allowscreation of channels within multiple print layers (different regionswithin z-axis) by printing the sacrificial polymer/hydrogel at thedesired print layers (heights).

FIG. 1 shows views outlining one embodiment of a printing method. Themethod involves 3D printing several layers of matrix material. A dualprint-head with light source 100 is utilized in the method. Thereference numeral 100 comprises a matrix ink print-head, a sacrificialink print-head and a light source as shown in FIG. 1. The portion ofreference numeral 100 (matrix ink print-head, sacrificial inkprint-head, and/or light source) that is closest to the shown layerindicates what portion is being utilized at that time during the method.A first matrix layer 101 is printed using a matrix ink and partiallycured for a certain period of time to form a partially cured matrixlayer 102. In one embodiment, the first matrix layer is partially curedusing a light source for around 10 seconds. The partial curing timecould vary depending on several factors, such as the material. A secondmatrix layer 103 is printed on the first matrix layer, which has beenpartially cured. A sacrificial material 104, such as a polymer orhydrogel, is printed using sacrificial ink within the second matrixlayer. In one embodiment, the sacrificial material is printed within thesecond matrix layer before the second matrix layer is exposed to light.The second matrix layer is sized to support the sacrificial material.The first matrix layer, the second matrix layer, and/or the sacrificialmaterial are partially cured as shown in 105 for a certain period oftime to crosslink the second matrix layer. In one embodiment, only thesecond matrix layer is exposed to light in this step. A third matrixlayer 106 is printed on the second matrix layer. The first matrix layer,the second matrix layer, the sacrificial material, and/or the thirdmatrix layer are partially cured as shown in 107 for a certain period oftime to crosslink the third matrix layer. In one embodiment, only thethird matrix layer is exposed to light in this step.

A 3D printed construct is formed, which includes the first matrix layer,the second matrix layer, the sacrificial material, and the third matrixlayer in this embodiment. While only three matrix layers and onesacrificial layer are shown in FIG. 1, the number of matrix layers andsacrificial layers could vary depending on the embodiment.

FIG. 2 are views outlining one embodiment of a post-printing method. Themethod involves curing of the 3D printed construct to crosslink thematrix polymer for a certain period of time. In one embodiment, the 3Dprinted construct is fully cured as shown in step 201 using a lightsource, and depending on the implementation, the time of cure is foraround 4 minutes. The curing time could vary depending on severalfactors, such as the material. The 3D printed construct is then immersedas shown in step 202 in an aqueous media to dissolve the sacrificialpolymer or hydrogel. Depending on the embodiment, any aqueous mediacould be used, such as PBS. The duration of immersion is determined bythe sacrificial material. In this particular case, the construct wasimmersed in PBS for 30 minutes at room temperature. It is possible toreduce this time significantly (about 5 min) when immersed at 4° C.Dissolution of the sacrificial layer leads to channels as shown in step203. While only two channels are shown in FIG. 2, depending on theembodiment that the number of channels could vary.

The materials and the methods of the present disclosure used in oneembodiment for a hydrogel scaffold and device will be described below.While the embodiment discusses the use of specific compounds andmaterials, it is understood that the present disclosure could employother suitable materials. Similar quantities or measurements may besubstituted without altering the method embodied below.

Methacrylated hyaluronic acid (MeHA) and alginate (MeAlg) MeHA hydrogelswere used as matrix bioinks. These polymers were synthesized asdescribed previously. Ink formulations were prepared by dissolving MeAlg(or MeHA) in PBS at different concentrations in the presence of aphotoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP),for blue light crosslinking. A blue light initiator was used as the 3Dprinter has a built in blue light source. Several ink formulations weredeveloped by varying the MeAlg (or MeHA) concentration. One of thesuitable bioink formulations was 9 wt. % MeHA, allowing extrusion basedprinting of the solution. The present inventors were able to generatestruts (individual lines) as small as 100-microns in diameter.

Pluronic® (F-127), a common sacrificial bioink, was used as asacrificial hydrogel, to create channels within matrix hydrogels. Forthis purpose, a dual head bioplotter was used to print the sacrificialand matrix bioink (MeHA) sequentially, as described in FIG. 1. For bothmatrix materials, the present inventors found that the partial curingtime of 10 seconds was sufficient to create self-supporting printedstructures. To create channels, sacrificial hydrogel was directlyprinted within the freshly printed matrix hydrogel layer as described inFIG. 1. After printing was completed, the scaffold was furthercrosslinked for 4 minutes. The scaffold was then immersed in PBS todissolve the Pluronic gel. The scaffold was removed from the petri dish,and the PBS in the channels was removed by applying a gentle vacuum. PBSwith red food coloring was then injected into each channel using aneedle (34- to 27-gauge needle).

The approach is versatile and enables development of complex channelswith tunable shape and size within photocurable hydrogels, eitherindividual or interconnected. FIGS. 3A-3D and FIGS. 4A-4C demonstratesome of the 3D designs and corresponding 3D printed structures.

As shown in FIGS. 3A-3D a 3D design is accomplished. In particular forthis embodiment is CAD designs of the hydrogel scaffolds with channels(light gray: matrix; dark gray: sacrificial). FIGS. 3A-3D are pictorialrepresentations showing the printed scaffolds after removal of thesacrificial regions. FIG. 3A illustrates a plurality of channels withvarying channel diameter. Depending on the implementation the diameterof the channels may all be the same or may be varied as represented inthis figure. FIG. 3B shows two-layer channel structure where channelsare in different planes. FIG. 3C illustrates a plurality ofinterconnected elliptical channels. Depending on the implementation thechannels may be different geometrical shapes or all the same shape. FIG.3D shows channels forming the logo NJIT. Coarse channels (500 μm indiameter) are printed for visual clarity and easy access with a regularsize needle, but 100 μm channels can be printed depending on theembodiment.

FIGS. 4A-4C show 3D printed structures, and feasibility studies. Asshown in FIG. 4A, a schematic of a channel design is illustrated. Thechannels may follow the same pathway or form various pathways as shownin this figure. FIG. 4B is a top view of an actual sample having thechannel design of FIG. 4A. FIG. 4B is a 3D printed construct withPluronic doped with red food coloring. FIG. 4C illustrates confocalimages of the channels in FIG. 4B taken at within the region of theblack rectangle in FIG. 4B and perfused with methacrylated rhodaminecontaining PBS, wherein the channels are smooth and cylindrical in shapewith width equal to about 500 μm.

FIG. 5 is a flow chart illustrating steps of one embodiment of thepresent method. Shown is one method for making a 3D scaffold. Step 501illustrates dual printing a matrix material up to a specific thicknessfor forming a printed layer. Step 502 shows exposing the matrix materialto a light source after each printed layer, and partially crosslinkingthe printed layer to allow self-supporting of the matrix material. Asshown in Step 503, printing a sacrificial layer with a sacrificialmaterial directly within an interface layer of the printed matrixmaterial is accomplished. Step 504 illustrates exposing the sacrificiallayer to the light source after printing of the sacrificial material.Step 505 illustrates printing a new layer of matrix material. Step 506shows how repeating the above first through fifth steps (501-505) may bedone to create a scaffold in a desired thickness or a human scale. Step507 illustrates exposing the scaffold to the light source to fullycrosslink the scaffold after printing and the desired thickness or thehuman scale is achieved. In Step 508, it is shown that immersing thescaffold into an aqueous solution is done to remove the sacrificialmaterial for a formation of channels.

While exemplary embodiments have been described herein, it is expresslynoted that these embodiments should not be construed as limiting, butrather that additions and modifications to what is expressly describedherein also are included within the scope of the invention. Moreover, itis to be understood that the features of the various embodimentsdescribed herein are not mutually exclusive and can exist in variouscombinations and permutations, even if such combinations or permutationsare not made express herein, without departing from the spirit and scopeof the invention.

What is claimed is:
 1. A method for making a 3D scaffold or a device,comprises: printing a photocurable polymer matrix material layer and asacrificial polymer material layer; and wherein the sacrificial polymermaterial layer is printed directly within the freshly printedphotocurable matrix material layer.
 2. The method of claim 1 furthercomprises: placing an extrusion needle only within an interface layerthat is partially crosslinked for free motion movement of the needlewithout using a shear thinning material; and wherein the printing isdone sequentially.
 3. The method of claim 1, wherein the matrix materialis a photo-curable material without shear thinning behavior.
 4. Themethod of claim 1, wherein the printing further comprises creating avascularized scaffold in a single step using dual extrusion printing. 5.The method of claim 4, wherein the vascularized scaffold is a hydrogelwith embedded channels for vascularization.
 6. The method of claim 4,wherein the vascularized scaffold is a soft microfluidic device madefrom an elastomer or a hydrogel.
 7. A method for making a 3D scaffold,comprises: directly and sequentially printing a photocurable matrixmaterial and a photocurable sacrificial material in a single step by adual extrusion based printing; fabricating a vascularized scaffold; andwherein the photocurable matrix material and sacrificial material is nota shear thinning material, and the sequential printing of thesacrificial material is within an interface of the matrix material. 8.The method of claim 7, wherein the vascularized scaffold is a device. 9.The method of claim 7, wherein the fabricating further comprises:forming an embedded channel using the dual extrusion based printing; andimplementing the sequential printing of the sacrificial material withinan interface layer of the photocurable matrix material after the matrixmaterial is partially cured by exposure to a light source.
 10. Themethod of claim 9, wherein the implementing step further comprisesplacing an extrusion needle only within the interface layer for freemotion movement of an extrusion needle without using shear thinningbehavior material for the matrix material.
 11. A method for making a 3Dscaffold, comprises: dual printing print a matrix material up to aspecific thickness for forming a printed layer; exposing the matrixmaterial to a light source after each of the printed layer, andpartially crosslinking the printed layer to allow self-supporting of thematrix material; printing a sacrificial layer with a sacrificialmaterial directly within an interface layer of the printed matrixmaterial; exposing the sacrificial layer to the light source afterprinting of the sacrificial material; printing a new layer of matrixmaterial; repeating above first through fifth steps to create a scaffoldin a desired thickness or a human scale; exposing the scaffold to thelight source to fully crosslink the scaffold after printing and thedesired thickness or the human scale is achieved; and immersing thescaffold into an aqueous solution to remove the sacrificial material fora formation of channels.
 12. The method of claim 11, wherein thethickness of the matrix material is manually determined by an end user.13. The method of claim 11, wherein the sacrificial layer is 100-1000microns thick depending on a desired channel size.
 14. The method ofclaim 11, wherein the aqueous solution is a phosphate buffer solution,(PBS).
 15. The method of claim 11, wherein the printing the sacrificiallayer further comprises placing an extrusion needle only within theinterface layer for a free motion movement of the needle without usingshear thinning behavior material.
 16. The method of claim 11, furthercomprises surface modifying a glass slide, and wherein the printing isdone on the surface modified glass slide or microscope cover slip foreasy handling of the scaffold.
 17. The method of claim 16, where thesurface modifying is done using 3-(trimethoxysilyl) propyl methacrylateto allow covalent cross linking of the matrix material to the glassslide.
 18. The method of claim 11 wherein the dual printing utilizes anextrusion-based printer system that allows extrusion of polymermaterials or solutions under applied pressure.
 19. The method of claim11, wherein the fabrication of channels is embedded in a hydrogel. 20.The method of claim 19, wherein the dual printing is a reservoir -freeprinting with no supportive hydrogel reservoir.